Peptides containing N-substituted D-amino acids for preventing β-strand association

ABSTRACT

Peptide is disclosed which comprises D-enantiomers of amino acids and is capable of interacting with other β-strand structure to form β-sheet, wherein said peptide is selectively Nα-substituted in one edge (first) of the β-strand-forming section of said peptide while the other edge (second) in the opposite orientation to the first edge in view of peptide backbone plane remains Nα-unsubstituted. Such the Nα-substituted peptide is capable of preventing association of said peptide with other β-strand (target) but permits interaction of said peptide with target β-strand in separate peptide-containing molecules through the Nα-unsubstituted edge. The peptide is useful for preventing β-strand association or aggregation.

This is a nationalization of PCT/GB00/02923, filed Jul. 28, 2000 andpublished in English.

The present invention relates to a class of peptide-based compounds thatbind specifically to target β-strands and thereby inhibit theirassociation into β-sheets and insoluble β-fibres. In particular, theinvention relates to peptides composed of D-enantiomers of amino acidsat least some of which are modified by Nα substitution.

A large number of terribly distressing, currently incurableneurodegenerative diseases are caused by the aggregation of proteins orpeptides into insoluble cytotoxic inclusions or amyloid-like plaqueswithin the brain: Alzheimer's disease (AD), which is the most commonform of senile dementia and the fourth most common cause of death in thedeveloped world, is caused by the aggregation of a 39–43-residue Aβpeptide fragment of a larger amyloid precursor protein (Forloni, 1996;Forloni et al., 1996; Joachim and Selkoe, 1992; Price et al., 1993;Selkoe, 1994; Verbeek et al., 1997; Wisniewski et al., 1997);Parkinson's disease (PD) and at least one form of dementia (Dementiawith Lewy Bodies, or DLB) are caused by the aggregation andincorporation of α-synuclein into intracytoplasmic inclusions calledLewy bodies (Arima et al., 1998; Baba et al., 1998; Mezey et al., 1998;Polymeropoulos, 1998; Spillantini et al., 1998; Trojanowski et al.,1998; Trojanowski and Lee, 1998); prion-related encephalopathies such asbovine spongiform encephalopathy (BSE, or ‘mad cow disease’) and itshuman forms Creutzfeldt-Jakob disease (CJD) and kuru are caused by theself-catalysed misfolding and aggregation of metastable proteins knownas prions (Forloni, 1996; Forloni et al., 1996; Horwich and Weissman,1997; Price et al., 1993; Prusiner and Dearmond, 1995); severaldominantly inherited neurodegenerative diseases including Huntington'sdisease (HD), X-linked spinal and bulbar muscular atrophy (SBMA),dentatorubral-pallidoluysian atrophy (DRPLA), and at least fivegenetically distinct forms of spinocerebellar ataxia (SCA types 1, 2, 3,6 and 7; SCA3 is better known as Machado-Joseph disease, or MJD) arecaused by the aggregation and incorporation of proteins or proteinfragments containing abnormally expanded glutamine repeats intointranuclear inclusions (Perutz, 1999; Ross, 1997).

In addition to these and undoubtedly many other, as yet unidentifiedneurodegenerative diseases, several non-neurodegenerative but equallydistressing diseases are caused by the aggregation of proteins orpeptides in other parts of the body. For example: type II diabetesmellitus is caused by aggregation of the 37-residue islet amyloidpolypeptide (IAPP or amylin) within the islets of Langerhans in thepancreas (Clark et al., 1996; Kahn et al., 1999; Obrien et al., 1993);familial amyloid polyneuropathy and senile systemic amyloidosis arecaused by the aggregation of full-length transthyretin and fragmentsthereof (Benson and Uemichi, 1996); and dialysis-related amyloidosis iscaused by the aggregation of β₂-microglobulin (Miyata et al., 1998).

In all these diseases, which are collectively known as amyloidoses, theproteins or peptides involved aggregate into insoluble β-fibres by theintermolecular association of β-strands into extended β-sheets; theseβ-fibres are deposited in inclusions or amyloid-like plaques which bringabout progressive cell death by some unknown mechanism. For more generalreviews on the amyloidoses and their mechanisms, see references(Kakizuka, 1998; Kisilevsky and Fraser, 1997; Serpell et al., 1997;Sunde and Blake, 1998; Wisniewski et al., 1998).

Although it remains to be determined how the aggregation of peptides andproteins into insoluble inclusions results in the progressive death ofcells, it is clear that the most effective general way to treat thediseases would be to prevent the formation of these cytotoxic inclusionsusing some agent that specifically inhibits the aggregation of proteinsand peptides into insoluble β-fibres. For one reason or another,however, none of the existing inhibitors of protein and peptideaggregation are suitable for use as therapeutic agents:

-   1) Simple organic compounds that act as protein denaturants such as    guanidinium chloride, urea, detergents, and many organic solvents    are very effective inhibitors of protein and peptide aggregation.    However, they tend to destabilise correctly folded proteins and    disrupt sensitive protein—protein interactions within the cell at    working concentrations because they are too simple in form to    inhibit protein and peptide aggregation specifically. As a    consequence they are toxic to cells, and are therefore unsuitable    for use as therapeutic agents.-   2) A number of more complex organic compounds have been found to    inhibit protein and peptide aggregation somewhat more specifically.    They include: β-cyclodextrin (Camilleri et al., 1994), congo red and    other sulphonated dyes (Burgevin et al., 1994; Lorenzo and Yankner,    1994; Pollack et al., 1995), nicotine (Salomon et al., 1996), hemin    and related porphyrins (Howlett et al., 1997), anthracycline    4′-iodo-4′-deoxydoxorubicin (Merlini et al., 1995),    hexadecyl-N-methylpiperidinium bromide (Wood et al., 1996),    melatonin (Pappolla et al., 1998), and rifampicin (Tomiyama et al.,    1994). None of these compounds have been found to be suitable for    use as therapeutic agents, however, therefore they are best regarded    as structural hits in the search for more active and    pharmacologically useful compounds. For a review on these compounds    as inhibitors of Aβ peptide aggregation, see reference (Bandiera et    al., 1997).-   3) Large proteins such as chaperonins or heat shock proteins (Kudva    et al., 1997), α2-macroglobulin (Hughes et al., 1998), laminin    (Bronfman et al., 1996), and monoclonal antibodies (Hanan and    Solomon, 1996; Solomon et al., 1996) can be extremely effective and    specific as inhibitors of protein and peptide aggregation because of    their size and complexity. However, they are too large to penetrate    cell membranes and the blood-brain barrier, they are susceptible to    aggregation and proteolysis, and they tend to be immunogenic.-   4) Simple peptides can also inhibit protein and peptide aggregation    effectively and specifically, and they are at least small enough to    penetrate cell membranes and the blood-brain barrier, which also    makes them less likely to be immunogenic than large proteins.    However, there is currently a conflict between the solubility,    hydrophobicity, and potency of these peptides, as well as a problem    of proteolytic degradation:

In Alzheimer's disease, for example, the 39–43-residue Aβ peptideaggregates into amyloid fibrils by the intermolecular association offive-residue peptide segments comprising the sequence KLVFF (SEQ IDNO: 1) (corresponding to residues 16–20 of the Aβ peptide) (Tjernberg etal., 1997; Tjernberg et al., 1996). The peptide segments form β-strandswhich associate to form an extended antiparallel β-sheet by means ofhydrophobic interactions between their side chains and hydrogen bondsbetween their backbone amide groups. This fibrogenic association can beinhibited by short peptides which also contain the KLVFF sequence (SEQID NO: 1) or a homologous sequence, such as Ac-QKLVFF-NH₂ (Tjernberg etal., 1996), GQKLVFFAEDVGG-[NH(CH₂)₅CO]-K₆ (Ghanta et al., 1996), andKKLVFFA (SEQ ID NO: 4) (Tjernberg et al., 1997). These peptides formβ-strands which compete for association with the homologous sequence inthe Aβ peptide and thereby hinder its aggregation. The first of thesepeptides has a limited solubility in aqueous solutions because it toocan aggregate into extended β-sheets. The latter two peptides, on theother hand, are more water-soluble because they contain more polargroups, but are consequently too hydrophilic to penetrate cell membranesand the blood-brain barrier. Peptides can be made more soluble withoutcompromising their hydrophobicity by including proline residues ratherthan polar residues. For example, the peptides RDLPFFPVPID, LPFFPVD, andLPFFD have a similar degree of hydrophobicity as the Aβ peptide, but arehighly soluble in aqueous solutions because the proline residuessterically prevent them from forming β-strands which aggregate intoextended β-sheets (Soto et al., 1996; Soto et al., 1998). However, thesepeptides are less potent inhibitors of Aβ-peptide aggregation becausethe β-strand conformation is actually required for making strong andspecific interactions with the β-strands formed by the Aβ peptide, inorder to inhibit their aggregation. In short, nobody has discovered howto prevent the peptides from aggregating in aqueous buffers withoutcompromising their hydrophobicity, which is required for effectivepenetration of cell membranes and the blood-brain barrier, or theirpotency as inhibitors of protein and peptide aggregation.

In addition to this problem of solubility versus hydrophobicity andpotency, all the peptides mentioned above are extremely susceptible todegradation by proteolytic enzymes because they consist entirely ofNα-unsubstituted α-L-amino acid residues, and are therefore unsuitablefor use as therapeutic agents. This particular problem has beenaddressed by designing peptides that consist only of α-D-amino acidresidues, which are not recognised by proteolytic enzymes (Miller etal., 1995). For example, all-D-[RDLPFFPVPID] (Soto et al., 1996) andall-D-[LFLRR] (Tjernberg et al., 1997) are highly resistant toenzyme-catalysed proteolysis as expected, but these peptides still facethe problem of conflict between solubility in aqueous buffers, abilityto penetrate cell membranes and the blood-brain barrier, and ability toinhibit the aggregation of other proteins and peptides into insolubleβ-fibres.

It is known that peptides containing Nα-substituted or α-D-amino acidresidues are much less susceptible to enzyme-catalysed proteolysis thanpeptides which consist only of Nα-unsubstituted α-L-amino acid residuesbecause neither Nα-substituted nor α-D-amino acid residues are notrecognised by proteolytic enzymes (Miller et al., 1995). Peptidescontaining Nα-substituted amino acid residues are also much less likelyto aggregate into insoluble β-fibres in aqueous solutions because the Nαatoms of these residues are not available for hydrogen bonding and,moreover, because their Nα substituents sterically disallow theassociation of β-strands. A peptide has been designed containingNα-methyl amino acid residues which folds into a three-stranded β-sheet,but which does not aggregate into extended β-sheets because theNα-methyl groups of these residues sterically prevent it from doing so(Doig, 1997). In this peptide, the two peripheral β-strands each containa sequence of two Nα-methyl alanine residues separated by a singleNα-unsubstituted alanine residue, so that all four Nα-methyl groups liealong the outer edges of these two β-strands, while the inner edgesremain free to associate with the central β-strand, thereby forming thethree-stranded β-sheet. However, it has not previously been reportedthat such a peptide is, in isolation, able to associate with β-strandsformed by other protein or peptide molecules and thereby inhibit theiraggregation into extended β-sheets and insoluble β-fibres. Moreover, ithas not previously been reported that a peptide comprisingNα-substituted and Nα-unsubstituted α-D-amino acid residues is able toassociate specifically with β-strands formed by other protein or peptidemolecules and thereby inhibit their aggregation into extended β-sheetsand insoluble β-fibres.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, therefore,there is provided a chemical compound or composition comprising apeptide, which peptide comprises a β-strand-forming section of peptidewhich forms a β-strand and associates as such with a target β-strandformed by a separate peptide-containing molecule, or comprising acomponent which mimics the structure and action of said β-strand-formingsection of peptide, wherein the β-strand-forming section of peptidecomprises a sequence of at least four consecutive α-D-amino acidresidues, all of which sterically permit the β-strand-forming section ofpeptide to form a β-strand, and at least one of which is anNα-substituted α-D-amino acid residue, and any two successiveNα-substituted α-D-amino acid residues are separated by an odd number ofconsecutive Nα-unsubstituted α-D-amino acid residues.

A β-strand is a section of peptide whose backbone takes on the form ofan extended ribbon; the side chains of consecutive residues in aβ-strand protrude from alternate sides of the plane of the ribbon, whilethe NH and CO components of the backbone peptide groups lie along thetwo edges of the ribbon. β-strands are regular structures that are onlyformed by sections of peptide which consist solely of a L aminoα-L-amino acid residues or solely of α-D-amino acid residues; the phiand psi angles of each amino-acid residue in a β-strand are close to−120° and +120° respectively. β-strands are not stable in isolation, andexist only when two or more of them are associated to form a parallel orantiparallel β-sheet. The individual β-strands in a β-sheet are heldtogether side by side and edge to edge in either parallel orantiparallel orientation by hydrogen bonds between the NH and COcomponents of their backbone peptide groups, as well as by additionalnon-covalent interactions between their side chains. A β-strand has twoedges, each of which can support the association of another β-strand inthis way. A β-sheet can therefore be extended indefinitely by theprogressive addition of more β-strands to the free edges of its twoperipheral β-strands; this eventually results in the formation ofinsoluble β-fibres.

The mechanism by which β-strands formed by proteins and peptidesaggregate into β-sheets and thereby insoluble β-fibres is illustratedschematically in FIG. 1. The peptides according to the invention inhibitthe aggregation of proteins and peptides into insoluble β-fibres bybinding specifically to the free edges of β-strands, thereby stericallyhindering their association into extended β-sheets. The entire peptidemay be involved in the formation of a β-strand, or only a sectionthereof, as referred to above. Where only a section of the peptide isinvolved in β-strand formation, it may be referred to as the“β-strand-forming section”.

A “section”, as referred to herein, is any part of an entity such as apeptide. Thus, when applied to peptides, “section” refers to a sequenceof contiguous amino-acid residues within, or at one end of, the peptide.The length of a “section” of peptide will depend upon the desiredapplication to which the section is to be put; for example,β-strand-forming section of peptide may be at least four amino-acids inlength, preferably longer, as set out below. The section may encompassthe whole of the peptide, or any part thereof. For example, it mayencompass 10%, 25%, 50%; 75%, 90% or 100% of the peptide.

“Successive”, as used herein, refers to any two defined amino-acidresidues which follow one another in a sequence, whether or not they arecontiguous in sequence. Thus, two successive Nα-substituted amino-acidresidues may be separated, if they are separated, by one or moreNα-unsubstituted amino-acid residues.

“Consecutive”, as used herein, refers to any two defined amino-acidresidues which follow one another in contiguous sequence. Thus, twoconsecutive Nα-unsubstituted amino-acid residues are adjacent in anamino-acid sequence.

According to the present invention, the chemical compound or compositionis separate from the target. The target is thus a discrete molecule,which either is a peptide or comprises a peptide. The target moleculemay thus be a peptide, a protein comprising a β-sheet peptide or sectionof peptide, a derivative of a protein, or any other molecule which iscapable of forming at least one β-strand.

The invention moreover relates to chemical compounds and compositionscomprising components which mimic the structure and action of a β-strandand are thus peptide mimics. A “peptide mimic” refers to a peptidewherein one or more of the backbone peptide groups or side-chain groupshave been replaced by another chemical group of similar stereochemistryand ability to form favourable non-covalent interactions with the targetβ-strand. For example, each backbone peptide group (CONH) could bereplaced by one of the following groups: CSNH (thioamide); COO (ester);CSO/(thioester); CSS (dithioester); COCH₂ (ketone); CSCH₂ (thioketone);SO₂NH (sulphonamide); SOCH₂ (sulphoxide); SO₂CH₂ (sulphone); SO₂O(sulphonate). Each N-substituted backbone peptide group could bereplaced by an N- or C-substituted form of one of the following groups:CSNH (thioamide); COCH₂ (ketone); CSCH₂ (thioketone); SO₂NH(sulphonamide); SOCH₂ (sulphoxide); SO₂CH₂ (sulphone). And each sidechain could be replaced by another group having a similarstereochemistry or arrangement of polar and non-polar atoms, as long asany particular features which are essential for association with thetarget β-strand are preserved.

The use of Nα-substituted α-D-amino acid is highly advantageous. Allα-D-amino acid are resistant to protease attack, and Nα-substitutedα-D-amino acid are also suitable for sterically hindering β-sheetformation. Resistance to protease attack is a preferred property in thecontext of the present invention.

In a second aspect of the present invention, there is provided a methodfor inhibiting or reversing the association of a target β-strand into aβ-sheet or β-fibre, comprising exposing the target β-strand to achemical compound or composition according to the first aspect of theinvention and allowing or inducing the chemical compound or compositionto associate with the target β-strand.

Optionally, in the method according to the preceding aspect of theinvention, other agents capable of destabilising β-sheet formation maybe used together with the peptides of the invention. For example, invitro use of a peptide according to the invention and a chaotrope, suchas Guanidiniun hydrochloride, is effective in preventing aggregation ofβ-strands to from β-sheets in solution.

In a third aspect, there is provided a method for inhibiting orreversing the aggregation of proteins or peptides, comprising contactingthe proteins or peptides with a chemical compound or compositionaccording to the first aspect of the invention.

In a fourth aspect, the invention provides a method for assisting in therefolding of denatured or aggregated proteins or peptides, comprisingcontacting the aggregated proteins or peptides with a chemical compoundor composition according to the first aspect of the invention.

In a fifth aspect, there is provided a chemical compound or compositionaccording to the first aspect of the invention for use in medicine.

In a sixth aspect, there is provided the use of a chemical compound orcomposition according to the first aspect of the invention for thepreparation of a composition for the diagnosis, study, or treatment of adisease caused by the aggregation of proteins or peptides into insolubleβ-fibres.

In a seventh aspect, the invention provides a method for inhibiting theoligomerisation or association of protein subunits, comprising exposingthe protein subunits to a chemical compound or composition according tothe first aspect of the invention.

The method of the seventh aspect may be applied, for example, to theinhibition of an enzyme whose catalytic activity depends on itsoligomerisation by the association of β-strands, either in vitro or invivo.

In an eighth aspect, there is provided a method for indicating thepresence or location of β-strands, β-sheets, or β-fibres, comprisingexposing a test sample to a chemical compound or composition accordingto the first aspect of the invention which comprises a detectablemoiety, removing any unbound chemical compound or composition, andassessing the test sample for the presence of the detectable moiety.

The test sample may be a histological sample and the chemical compoundor composition may be used as a histochemical stain or indicator.

In a ninth aspect, the invention relates to a method for affinity orprotein-renaturation chromatography, comprising the steps of covalentlyattaching a chemical compound or composition according to the firstaspect of the invention to a solid matrix, resin, or support; passing atest sample over the column; and separating the desired treated productfrom the column.

In a tenth aspect, the invention provides a combinatorial librarycomprising chemical compounds or compositions according to the firstaspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates schematically how β-strands associate into extendedβ-sheets and thereby insoluble β-fibres. In this figure, the β-strandsare represented by white jigsaw pieces which have both a circular taband a circular hole of the same size on each of their two long edges,representing the CO and NH components of the backbone amide groups alongthe two edges of the β-strands. The β-strands may associate in eitherthe parallel or antiparallel orientation into extended β-sheets andinsoluble β-fibres by the formation of hydrogen bonds between these COand NH components; in FIG. 1 this interaction is represented by themutual insertion of circular tabs into the circular holes of associatedjigsaw pieces, which may lead to the production of extended chains ofjigsaw pieces representing the extended β-sheets and insoluble β-fibres.

FIG. 2 illustrates schematically how β-strands formed by theβ-strand-forming sections of peptide in the peptides of the inventionare able to inhibit the aggregation of target β-strands formed by otherproteins and peptide molecules into extended β-strands and insolubleβ-fibres. In this figure, the target β-strands are represented by whitejigsaw pieces which have both a circular tab and a circular hole of thesame size on each of their two long edges, just as they are in FIG. 1,while the β-strands formed by the β-strand-forming sections of peptideare represented by shaded jigsaw pieces. One edge of these shaded jigsawpieces has both a circular tab and a circular hole, representing the COand NH components of the backbone amide groups which lie along the freeedge of the β-strand formed by the β-strand-forming is section ofpeptide. This edge of the shaded jigsaw pieces is identical to bothedges of the white jigsaw pieces, and can therefore be joined to thewhite jigsaw pieces in either parallel or antiparallel orientation justas the white jigsaw pieces are able to be joined to each other. Theother edge of the shaded jigsaw pieces, however, has two circular tabsbut no circular hole, representing the fact that one, some, or all ofthe backbone NH groups along one edge of the β-strand formed by theβ-strand-forming section of peptide are sterically blocked by theNα-substituents which lie along it. This edge of the shaded jigsawpieces is consequently unable to be joined to any other jigsaw piece,whether it be white or shaded. Therefore, when the single-tab edge of ashaded jigsaw piece is joined in either the parallel or antiparallelorientation to either single-tab edge of a white jigsaw piece, which mayor may not form part of a longer chain of such pieces, no other jigsawpiece may subsequently be joined to that edge of the white jigsaw piece,and elongation of a chain in that direction is thereby blocked as shown,unless the terminal shaded jigsaw piece is first removed. In just thesame way, the β-strands formed by the β-strand-forming section ofpeptide bind to the target β-strands formed by other protein and peptidemolecules and thereby inhibit their aggregation into extended β-strandsand insoluble β-fibres. This jigsaw model thus clearly illustrates thefundamental concept of the present invention.

FIGS. 3 and 4 show how Peptide X SEQ ID NO: 2) forms a β-strand (X) andassociates as such with one edge of a target β-strand (Y) formed by asegment of the Aβ peptide or some other peptide-based molecule in eitherorientation to form a parallel (FIG. 3) or antiparallel (FIG. 4)two-stranded β-sheet complex, thereby sterically hindering theassociation of other β-strands with that edge of the target β-strand. Ineach of these two figures: the target β-strand comprises a sequence ofeight consecutive α-L-amino acid residues, the Cα atoms of which havenot been labelled; the Cα atoms of the six α-D-amino acid residues ofPeptide X SEQ ID NO: 2) are numbered from the N-terminus, while the Cαatom of its N-terminal acetyl group is indicated by a letter A; only thenon-hydrogen backbone atoms of these two β-strands—including theNα-methyl carbon atoms of the two Nα-methyl-α-D-amino acid residues(residues 2 and 4) of Peptide X SEQ ID NO: 2)—are shown, and arerepresented by symbols defined by the atom key below the figures;hydrogen bonds between backbone amide groups of the two β-strands areindicated by dashed lines.

FIG. 5 is a graph showing the prevention of Alzheimer's Aβ peptideaggregation into β-sheet structures after administration of Peptide XSEQ ID NO: 2). A 50% reduction in Alzheimer's Aβ peptide aggregation isseen at a Peptide X SEQ ID NO: 2) concentration of 100 mM.

FIG. 6 is an electron micrograph showing aggregated Alzheimer's Aβpeptides. Alzheimer's Aβ peptide was incubated at a concentration of 500mM and the aggregate examined by electron microscopy.

FIG. 7 is an electron micrograph showing Alzheimer's Aβ peptidesincubated at a concentration of 500 mM in the presence of Peptide X SEQID NO: 2); electron microscope examination shows a substantialelimination of aggregation.

DETAILED DESCRIPTION OF THE INVENTION

The peptides according to the invention inhibit the aggregation ofproteins and peptides into insoluble β-fibres by binding specifically tothe free edges of β-strands, thereby sterically hindering theirassociation into extended β-sheets. They do this substantially asfollows:

The peptide according to the present invention comprises a section whichis able to form a β-strand, because it consists solely of α-D-amino acidresidues which sterically permit it to do so. On top of this, the stericconstraints imposed by the Nα-substituted α-D-amino acid residue(s) andby any β-branched α-D-amino acid residue(s) in the β-strand-formingsection of peptide may serve to encourage β-strand formation. When theβ-strand-forming section of peptide forms a β-strand, theNα-substituents of its Nα-substituted α-D-amino acid residues arepositioned, by design, so as to lie along only one of its two edges. TheNα-substituted residues are spaced such that they are separated by oddnumbers of residues, since the repeating unit of a β-strand is tworesidues. For example, between any two successive Nα-substitutedresidues there may lie 1 or 3 Nα-unsubstituted residues.

The Nα-substituted edge of the β-strand is unable to associate withother β-strands formed by the β-strand forming section of peptidebecause the Nα-substituents which lie along it sterically prevent itfrom doing so. The other, free edge of this β-strand is able to do so,and may associate in either the parallel or antiparallel orientationwith a free edge of a target β-strand formed by another protein orpeptide molecule by means of hydrogen bonds between their backbonepeptide groups and additional non-covalent interactions between theirside chains. This target β-strand is most likely to be one of the twoperipheral β-strands of an existing β-sheet, but could also be a single,isolated β-strand that forms only as it associates with the β-strandformed by the β-strand-forming section of peptide. Either way, theresult of this association is the formation of a β-sheet complex whereinthe β-strand formed by the β-strand-forming section of peptidesterically blocks the association of other β-strands with the nowassociated edge of the target β-strand, thereby preventing the formationof an extended β-sheet and the deposition of insoluble β-fibres. Forexample, if the target β-strand is one of the two peripheral β-strandsof an existing β-sheet, then the association of the β-strand formed bythe β-strand-forming section of peptide with the free edge of thattarget β-strand sterically blocks the association of other β-strandswith that edge of the target β-strand, thereby preventing extension ofthe β-sheet in that direction. Extension of the β-sheet in the otherdirection may be prevented in the same way by association of theβ-strand formed by the β-strand-forming section of peptide with the freeedge of the other peripheral β-strand of the β-sheet. Isolated targetβ-strands may be prevented from associating with each other bysimultaneous association of both edges with two β-strands formed by theβ-strand-forming section of peptide. In this case, the resultingthree-stranded β-sheets can not be extended in either direction due tosteric hindrance by the Nα substituents which lie along the outer edgesof both the peripheral β-strands.

As used herein, a “peptide” is a polymer in which the monomers are aminoacids and are joined together by peptide bonds. The length of aβ-strand-forming section of peptide according to the invention will bedetermined empirically, as described in detail below; however, theβ-strand-forming section of peptide is at least 4 amino acid residues inlength, and preferably between about 4 and about 50 amino acid residuesin length; advantageously between about 4 and about 16 amino acidresidues in length, and most preferably between about 5 and about 10amino acid residues. Preferably, the β-strand-forming section of peptideis no longer than the target β-strand, and at least as long as theaggregation-causing section of the target β-strand.

The amino-acid monomers of which the β-strand-forming section of peptideis constructed are α-D-amino acids, meaning that they are of theD-enantiomeric from as opposed to the L-enantiomeric form. D-aminoacids, which commonly occur in nature, are susceptible to digestion byprotease enzymes if unprotected. Nα-substituted α-D-amino acids areα-D-α-D-amino acids which carry a substituent, which is not hydrogen, onthe α-N atom, whilst Nα-unsubstituted α-D-amino acids have nosubstituent at this position. Preferred substituents useful forpractising the subject invention are set forth below. In general,however, the substituents must be large enough to sterically hinder theassociation of β-strands, and preferably large enough to hinder orprevent proteolytic degradation of the peptide but they must not hinderthe β-strand forming section of peptide from forming a β-strand.

As used herein, “destabilising”, when applied to β-sheets and β-sheetformation, refers to the inhibition of β-strand aggregation into β-sheetstructures and preferably the prevention of β-strand aggregation.Advantageously, it refers to the reversal of β-strand aggregation andactual disruption of β-sheet structures. Reversal may be complete orpartial; in general, reversal indicates that p-sheet structures revertto unassociated β-strands, or are split up into smaller β-sheets.“Hinder”, “inhibit” and “prevent”, as used above, refer to a reductionin β-strand aggregation ranging from partial to substantially complete.For example, β-strand aggregation may be reduced by 20%, 30%, 50%, 75%or more, preferably about 90% or 100%.

The side chains used in β-strand-forming sections of peptide accordingto the invention moreover allow or favour the formation of β-strands.“Allow”, as used herein, means that the formation of β-strands is notimpeded. “Favour” means that such formation is facilitated with respectto any selected amino-acid which merely allows β-strand formation.

The concept of favouring or allowing β-strand formation may be expressedin terms of β-sheet propensity values for amino-acid residues. β-sheetpropensity is a measure of the incidence of particular amino-acids inβ-sheets formed by natural proteins; it has been found that thepropensity value correlates very well with the thermodynamicconsiderations which govern β-sheet formation by amino-acid residues.See, for example, Williams et al., (1987); Wilmot and Thornton, (1988);Kim and Berg, (1993); Smith et al., (1994); Minor and Kim, (1994a);Regan, (1994); and Bai and Englander, (1994). Advantageously, residuesincorporated into the β-strand-forming section of peptide have a β-sheetpropensity of at least about 1.00.

Design of Peptides According to the Invention

In order that the β-strand-forming section of peptide is able to form aβ-strand, it must consist solely of α-D-amino acid residues whichsterically permit the β-strand-forming section of peptide to form aβ-strand. Proline, for example, cannot be included in theβ-strand-forming section of peptide except at its very ends because itsside chain is joined back onto its backbone nitrogen atom, and thereforeit is unable to adopt the phi angle required to form a β-strand.

In order that the β-strand formed by the β-strand-forming section ofpeptide associates strongly enough with a target β-strand to inhibit itsaggregation into insoluble β-fibres, it must be at least four amino-acidresidues in length. A β-strand consisting of three or fewer amino-acidresidues would not interact with a target β-strand strongly enough tohinder the association of other β-strands with that target β-strand. Ingeneral, the β-strand-forming section of peptide may be any lengthgreater than three residues (i.e. four or more), but in practice shouldbe no longer than the target β-strand, and should preferably be at leastas long as the segment of that target β-strand which is directlyresponsible for its aggregation.

This is because the aggregation-causing segment of the target β-strandis likely to comprise a sequence of residues having hydrophobic oramide-containing side chains, which can form the strongest interactionswith the adjacent side chains of an associated β-strand in aqueoussolutions. It is this aggregation-causing segment of the target β-strandwith which the β-strand-forming section of peptide is preferablydesigned to associate. Whilst the β-strand-forming section of peptideaccording to the invention may be shorter, the same length or longerthan the aggregation-causing segment of the target β-strand, there is noneed for the β-strand forming section of peptide to be any longer thanthe target β-strand itself, because any additional residues in theβ-strand-forming section of peptide are unlikely to interact stronglywith the residues which flank the target β-strand, if such residues arenot in a β-strand structure.

The target β-strand, the aggregation-causing segment of that targetβ-strand and therefore the optimal length for the β-strand-formingsection of a peptide according to the present invention, may bedetermined empirically. For example, the target β-strand may beidentified as a section of peptide in a protein or peptide moleculewhich forms a β-strand and undesirably aggregates or associates as suchwith other β-strands to form a β-sheet or β-fibre. Theaggregation-causing segment of this target β-strand can then beidentified as a section of at least four residues mostly havinghydrophobic and/or amide-containing side chains, or can be determinedexperimentally by investigating the association properties of shortsegments of the target β-strand or of single-residue mutants of thetarget β-strand. For example, a section of the 39–43-residue Alzheimer'sAβ peptide forms a β-strand and undesirably aggregates as such intoinsoluble β-fibres. This β-strand is therefore identified as the targetβ-strand, and its aggregation-causing segment has been identified ashaving the sequence KLVFF SEQ ID NO: 1) by investigating the associationproperties of short segments of the Aβ peptide and single-residuemutants thereof: truncation of this segment at either end, orsubstitution of any of its residues by alanine dramatically reduced thetendency of the Aβ peptide to aggregate into insoluble β-fibres(Tjernberg et al., 1997; Tjernberg et al., 1996).

It will be appreciated by those skilled in the art that similarprocedures may be used to identify target β-strands in proteins otherthan Aβ, or to identify alternative target β-strands in Aβ, usingsimilar (or other) procedures, as known in the art and/or describedherein.

The β-strand-forming section of peptide according to the invention ispreferably designed to form a β-strand and associate as such in theparallel orientation with this aggregation-causing segment of the targetβ-strand to form a parallel β-sheet complex. Preferably, it is designedas follows.

The β-strand-forming section of peptide preferably contains the samenumber of residues as the aggregation-causing segment of the targetβ-strand, and advantageously comprises a sequence of alternatingNα-methyl-α-D-amino acids and Nα-unsubstituted α-D-amino acid residues.The side chains of the residues in the β-strand-forming section ofpeptide are complementary to those of the aggregation-causing segment ofthe target β-strand in the same order, by which is meant the side chainof the first residue of the β-strand-forming section of peptide ischosen to form a favourable non-covalent interaction with the side chainof the first residue of the aggregation-causing segment of the targetβ-strand, and so on. For example: if the first residue of theaggregation-causing segment of the target β-strand has anamide-containing side chain, then the first residue of theβ-strand-forming section of peptide should also have an amide-containingside chain; if the first residue of the aggregation-causing segment ofthe target β-strand has a hydrophobic side chain, then the first residueof the β-strand-forming section of peptide should also have ahydrophobic side chain; if the first residue of the aggregation-causingsegment of the target β-strand has a hydroxyl-containing side chain,then the first residue of the β-strand-forming section of peptide shouldalso have a hydroxyl-containing side chain; if the first residue of theaggregation-causing segment of the target β-strand has a basic sidechain, then the first residue of the β-strand-forming section of peptideshould have an acidic side chain; and if the first residue of theaggregation-causing segment of the target β-strand has an acidic sidechain, then the first residue of the β-strand-forming section of peptideshould have a basic side chain. This selection procedure is continuedfor all the remaining side chains in the β-strand-forming section ofpeptide.

In general, a suitable sequence of side chains in the β-strand-formingsection of peptide can also be taken directly from the section of theβ-strand which undesirably associates with the aggregation-causingsection of the target β-strand. For example, the Alzheimer's Aβ peptideaggregates into insoluble β-fibres by the intermolecular association ofidentical KLVFF (SEQ ID NO: 1) aggregation-causing segments of peptideas β-strands in the antiparallel orientation, and in the resultingantiparallel β-sheet complex, the four hydrophobic side chains of eachβ-strand form hydrophobic interactions with those of the associatedβ-strand, while the basic lysine side chain of each β-strand presumablyforms an electrostatic interaction with one of the two acidic sidechains that follow the KLVFF sequence (SEQ ID NO: 1) in the associatedβ-strand (Tjernberg et al., 1997). Since the β-strand-forming section ofpeptide is designed to associate as a β-strand with the KLVFF sequence(SEQ ID NO: 1) in the parallel orientation, the sequence of its sidechains is preferably designed to be homologous or identical to the KLVFFsequence (SEQ ID NO: 1) in reverse order, i.e. FFVLK (SEQ ID NO: 3.Other compounds or compositions corresponding to the present inventionmay be designed to associate specifically with other target β-strands bya similar method.

The de novo design of β-sheet polypeptides has been described in theart. For example, reference is made to Smith and Regan, (1995); Smithand Regan, (1997); De Alba et al., (1999); and Kortemme et al., (1998).These and other approaches may be employed in designing a suitablepolypeptide. For instance, a suitable sequence of side chains in theβ-strand-forming section of peptide may be determined by constructing amolecular model of a parallel or antiparallel β-sheet complex in whichthe target β-strand is associated with a second β-strand, and thenadapting the identity and conformation of the side chains of the secondβ-strand to make favourable non-covalent interactions with the sidechains of the target β-strand. This may be done using a computer andappropriate software as follows:

First, a molecular model of the target β-strand is constructed. This maybe done by extracting the coordinates of a β-strand in a protein ofknown molecular structure, and then changing the sequence of its sidechains to that of the target β-strand. Next, a molecular model of asecond β-strand is constructed by a similar method, is transformed intoits own mirror image and is then positioned alongside either edge of thetarget β-strand in the parallel or antiparallel orientation to form atwo-stranded parallel or antiparallel β-sheet complex. Possible sidechains for each consecutive residue in the second β-strand are thenconsidered, and their alternative conformations are explored todetermine whether they are likely to form favourable non-covalentinteractions with the neighbouring side chains of the associated targetβ-strand in the β-sheet complex. Finally, once a suitable sequence ofside chains is selected, energy-minimisation and molecular dynamicsprograms may be applied to investigate the theoretical validity of themodel, before synthesising the candidate peptide and testing itexperimentally for activity.

Other guidance relating to the design of peptides which form β-strandsmay be found in the foregoing material relating to β-sheet propensitiesfor amino-acids, as well as the following sources: Nelsoney and Kelly,(1996); Hutchinson et al., (1998); Pham et al., (1998); Minor and Kim,(1996); Koepf et al., (1999); and Minor and Kim, (1994b).

Selection and Location of Nα-Substituents

In order that the Nα-substituents of the Nα-substituted α-D-amino acidresidues in the β-strand-forming section of peptide lie along only oneof the two edges of the β-strand formed by the β-strand-forming sectionof peptide, the Nα-substituted α-D-amino acid residues are interspersedby odd numbers of unsubstituted amino-acids, unless there is only oneNα-substituted α-D-amino acid residue in the β-strand-forming section ofpeptide, because the repeating unit of a β-strand is two residues: ifany two Nα-substituted α-D-amino acid residues in the β-strand-formingsection of peptide were adjacent or separated by an even number ofunsubstituted residues, then their Nα substituents would lie on oppositeedges of the β-strand, and neither edge of the β-strand would be able toassociate with a target β-strand and thereby sterically hinder theassociation of other β-strands with that target β-strand.

In theory, therefore, the Nα-substituted α-D-amino acid residues in theβ-strand-forming section of peptide could be very large numbers ofresidues apart, or there could be only one Nα-substituted α-D-amino acidresidue in the β-strand-forming section of peptide. In practice,however, successive Nα-substituted α-D-amino acid residues in theβ-strand-forming section of peptide should preferably be separated by nomore than 3 unsubstituted residues because the steric constraintsimposed by these residues actually serve to encourage theβ-strand-forming section of peptide to adopt the active β-strandconformation (Manavalan and Momany, 1980).

In the most preferable case therefore, successive Nα-substituted aminoacid α-D-amino acid residues in the β-strand-forming section of peptideare separated from each other by single unsubstituted residues so thatthe β-strand-forming section of peptide comprises a sequence ofalternating Nα-substituted and Nα-unsubstituted α-D-amino acid residues.This induces the entire section of peptide to adopt an active β-strandconformation.

The Nα-substituents may be substantially any atom or group that islarger than a hydrogen atom, which essentially means any atom or groupother than a hydrogen atom. However, they must also not stericallyprevent the β-strand-forming section of peptide from forming a β-strand,because the β-strand-forming section of peptide has to form a β-strandin order to associate with a target β-strand.

The Nα-substituent is thus optionally a fluorine atom or a hydroxygroup, or another group that is connected to the Nα atom by an oxygenatom within it, such as a methoxy group or another alkoxy group.

Preferably, the Nα-substituent is a group that is connected to the Naatom by a methylene (CH₂) group within it. Such a group can beincorporated into the β-strand-forming section of peptide by standardmethods of solution- or solid-phase peptide synthesis, and theconnecting methylene group is sterically compatible with the formationof a β-strand. Suitable examples of this preferred form ofNα-substituent include a methyl or ethyl group, or another alkyl oraliphatic group that is connected to the Nα atom by a methylene (CH₂)group within it, or a substituted or unsubstituted benzyl group, such asan acetylated or otherwise acylated 2-hydroxy-4-methoxybenzyl (AcHmb)group, or another aryl-methyl group.

A methyl group is the most preferred form of Nα-substituent because itis the simplest group that can be incorporated into the β-strand-formingsection of peptide by standard methods of solution- or solid-phasepeptide synthesis, and the corresponding amino-acids and their Fmoc andBoc derivatives are commercially available.

The 2-hydroxy-4-methoxybenzyl (AcHmb) group is a further preferred formof Nα-substituent because the corresponding amino-acids and their Fmocand Boc derivatives are also commercially available, but this group isfairly labile unless its 2-hydroxyl group is acetylated or otherwiseacylated (Quibell et al., 1995; Quibell et al., 1995; Quibell et al.,1994).

The 2-hydroxybenzyl (AcHb) group is yet another preferred form ofNα-substituent, and unlike the 2-hydroxy-4-methoxybenzyl group, it doesnot need to be acetylated or otherwise acylated (Johnson and Quibell,1994).

The β-strand-forming propensity of the β-strand-forming section ofpeptide may be increased further by including Nα-substituted orNα-unsubstituted α-D-amino acid residues whose side chains stericallyfavour the β-strand conformation. These include α-D-amino acid residueswith β-branched side chains, such as α-D-threonine, α-D-valine,α-D-isoleucine, α-D-tert-leucine, α-D-p-hydroxyvaline, and theirNα-substituted derivatives. Other α-D-amino acid residues which favourthe β-strand conformation, for example those with aromatic side chainssuch as α-D-tyrosine, α-D-phenylalanine and α-D-tryptophan, and thosewith aliphatic hydrophobic side chains such as α-D-leucine andα-D-methionine, plus α-D-serine and α-D-glutamine, should also beincluded in the β-strand-forming section of peptide if and whereappropriate: they must be compatible with the chemistry of solution- orsolid-phase peptide synthesis, they must not sterically hinder theassociation of the free edge of the β-strand formed by theβ-strand-forming section of peptide with the target β-strand, and theyshould preferably promote the β-strand formed by the β-strand-formingsection of peptide to associate tightly with the target β-strand.

As explained above, the Nα-substituted and Nα-unsubstituted α-D-aminoacid residues in the β-strand-forming section of peptide shouldpreferably promote the β-strand-forming section of peptide to form aβ-strand; but they should also preferably promote this β-strand toassociate as tightly as possible with the target β-strand. For this,their side chains should form strong non-covalent interactions with theneighbouring side chains of the target β-strand when the two strands areassociated with each other in the parallel or antiparallel β-sheetcomplex. The strongest non-covalent interactions that can exist betweenthe neighbouring side chains of associated β-strands in aqueoussolutions are hydrophobic interactions between hydrophobic side chainsand hydrogen bonds between amide-containing side chains. The segment ofthe target β-strand most responsible for its aggregation is likely to berich in residues which have these side chains, and therefore it is thisaggregation-causing segment of the target β-strand with which theβ-strand-forming section of peptide can potentially associate mosttightly. For this reason, most of the Nα-substituted andNα-unsubstituted α-D-amino acid residues in the β-strand-forming sectionof peptide preferably have hydrophobic or amide-containing side chains.The preferred amino-acid residues with hydrophobic side chains includeα-D-valine, α-D-leucine, α-D-isoleucine, α-D-methionine,α-D-phenylalanine, α-D-tyrosine, α-D-tryptophan, and theirNα-substituted derivatives; while the preferred amino-acid residues withamide-containing side chains include α-D-asparagine, α-D-glutamine, andtheir Nα-substituted derivatives. The most preferred side chain of eachresidue in the β-strand-forming section of peptide depends on theneighbouring side chain of the associated target β-strand in the β-sheetcomplex, because their stereochemistries must be compatible with theformation of a favourable non-covalent interaction between them. Ingeneral, however, the most preferred hydrophobic side chain is that ofleucine because it is fairly large but relatively flexible, being ableto adopt any one of nine different rotamer conformations, and can easilyadapt its stereochemistry to make the most favourable hydrophobicinteraction with almost any neighbouring hydrophobic side chain of anassociated target β-strand; the most preferred amide-containing sidechain is that of glutamine because it too is relatively flexible, and ismore likely to be able to make a favourable hydrogen bond with aneighbouring glutamine or asparagine side chain of an associated targetβ-strand. However, any hydrophobic side chain, or side chain which has aconsiderable hydrophobic portion, could be included in theβ-strand-forming section of peptide, as could any amide-containing sidechain, as long as they did not sterically hinder the β-strand-formingsection of peptide from forming a β-strand, or from associating as suchwith a target β-strand.

Although most of the Nα-substituted and Nα-unsubstituted α-D-amino acidresidues in the β-strand-forming section of peptide should havehydrophobic or amide-containing side chains, the remainder of theNα-substituted and Nα-unsubstituted α-D-amino acid residues in theβ-strand-forming section of peptide may have side chains which areneither hydrophobic nor amide-containing, but which can neverthelessform favourable non-covalent interactions with the neighbouring sidechains of an associated β-strand. For example: the acidic side chains ofaspartate and glutamate may form salt bridges with the basic side chainsof histidine, arginine, and lysine in an associated β-strand, andconversely, the basic side chains of histidine, arginine, and lysine mayform salt bridges with the acidic side chains of aspartate and glutamatein an associated β-strand; the hydroxyl-containing side chains ofserine, threonine, and β-hydroxyvaline may form hydrogen bonds with theneighbouring hydroxyl-containing side chains of an associated β-strand.

Prevention of β-Sheet Stacking

In order that the β-sheets formed by association of the β-strands do notaggregate by stacking, the β-strand-forming section of peptide alsopreferably includes one or more α-D-amino acid residues having a sidechain which extends beyond the neighbouring side chains in the β-strandformed by the β-strand-forming section of peptide. Such an extended sidechain is preferably long and preferably has a polar end, so that it doesnot support the stacking of β-sheets. The side chains of lysine andarginine are suitable examples of such extended side chains having apolar end.

Labelling of Peptides

In order that the peptides according to the invention can be traced ordetected, the β-strand-forming section of peptide may include anα-D-amino acid residue having a side chain which contains a radioactiveor magnetically active nucleus, such as an α-D-phenylalanine,α-D-tyrosine, or α-D-thyronine residue with one or more radioactive ormagnetically active iodine or other halogen atoms substituted onto thearomatic ring(s); or the β-strand-forming section of peptide may includean α-D-amino acid residue having a side chain which contains afluorescent, coloured, or other spectroscopically detectable group,including spin labels such as the 2,2,5,5-tetramethyl-1-pyrrolidinyloxy(PROXYL) and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) groups whichcontain unpaired electrons. A peptide containing such aspectroscopically detectable group or a radioactive or magneticallyactive nucleus may be used as a traceable probe to indicate the presenceand location of target β-strands or insoluble β-fibres, either in vitroor in vivo.

Membrane Penetration

In order that the compound or composition can more easily penetrate cellmembranes and the blood-brain barrier, the β-strand-forming section ofpeptide preferably contains a high proportion of amino-acid residueshaving hydrophobic or basic side chains. The hydrophobic side chainsinteract with the hydrophobic portions of the phospholipid moleculeswhich constitute these barriers, while the basic side chains mightinteract with the phosphate head groups of these molecules, just as thebasic side chains in the membrane-penetrating peptide segments of theDrosophila Antennapedia homeodomain and the HIV-1 Tat protein have beenproposed to do (Derossi et al., 1996; Vives et al., 1997; Vives et al.,1997).

Alternatively, or in addition to the foregoing, the peptide of theinvention may be encouraged to penetrate cell membranes and theblood-brain barrier more easily by arranging the β-strand-formingsection of peptide such that it is preceded or followed in the peptidesequence by, or otherwise attached to, a distinct membrane-penetratingsection of peptide that consists entirely or almost entirely ofamino-acid residues having basic or hydrophobic side chains. Thesemembrane-penetrating sections of peptide are able to carry peptides andsmall proteins to which they are attached through cell membranes and theblood-brain barrier by interacting with the phospholipid molecules whichconstitute these biological barriers, as described above. Other sectionsof peptide which are rich in residues with basic and/or hydrophobic sidechains may also be able to act as vectors for carrying theβ-strand-forming section of peptide through these barriers (Derossi etal., 1998). The side chain of each residue in the membrane-penetratingsection of peptide is preferably a basic or hydrophobic group, such asthose of alanine, valine, leucine, isoleucine, methionine,phenylalanine, tyrosine, tryptophan, proline, histidine, lysine, andarginine. The membrane-penetrating section of peptide may also includeα-D- or Nα-substituted amino-acid residues to make it more resistant toenzyme-catalysed proteolytic degradation.

The membrane-penetrating section of peptide may be attached to theβ-strand-forming section of peptide by including it in the solid-phasesynthesis of the β-strand-forming section of peptide as one continuouspeptide, wherein the membrane-penetrating section of peptide eitherprecedes or follows the β-strand-forming section of peptide.Alternatively, the membrane-penetrating section of peptide may beattached via an amide or disulphide bond to one of the side chains ofthe β-strand-forming section of peptide.

The β-strand-forming section of peptide may have a free, acetylated, orotherwise acylated N terminus and/or a free, amidated, or esterified Cterminus, or may form part of a larger peptide which has a free,acetylated, or otherwise acylated N terminus and/or a free, amidated, oresterified C terminus. Amidation or esterification of the C terminus ispreferable because a free carboxyl group reduces the ability of apeptide to penetrate cell membranes and the blood brain barrier, due tounfavourable electrostatic interactions between this negatively chargedgroup and the negatively charged phosphate head groups of thephospholipid molecules which constitute these barriers.

Acetylation or acylation of the N-terminal amino group may actuallyreduce the ability of the peptide to penetrate cell membranes and theblood brain barrier because a free positively charged N-terminal aminogroup would form favourable electrostatic interactions with thenegatively charged phosphate head groups of the phospholipid molecules,and thereby help the peptide to cross these barriers.

However, a free positively charged N-terminal amino group would not formas strong a hydrogen bond with the backbone carbonyl oxygen atom of anassociated target β-strand as an acetylated or otherwise acylatedN-terminal amino group would. Therefore, an acetylated or otherwiseacylated N-terminal amino group is preferred if the N-terminal aminogroup forms part of the β-strand-forming section of peptide: the problemof reduced ability of the peptide to penetrate cell membranes and theblood-brain barrier can be overcome by attaching residues with basicside chains or a distinct membrane-penetrating section of peptide toeither end of the β-strand-forming section of peptide, as describedabove.

Attachment of Functional Groups

The peptide according to the invention may be attached to a functionalcomponent. This functional component may be a section of peptide orother molecule which causes the compound or composition to targetspecific organs, cells, or molecules, such as a hormone, antibody,transcription factor, or other protein molecule; or it may be a label asdescribed above, such as an atom or group that contains a radioactive ormagnetically active nucleus; or it could be a fluorescent, coloured, orother spectroscopically detectable group; or it could be a group whichcontains an unpaired electron and thereby acts as a spin label, such asthe 2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL) group or the2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) group; or it may be anenzyme, or a cytotoxic molecule which selectively kills cells containingor otherwise associated with the target β-strand; or it may be a solidmatrix, resin, or support.

The β-strand-forming section of peptide is attached to any of thesefunctional components or some other functional component by means of anamide bond, ester bond, or any other suitable linkage between a sidechain, Nα-substituent, or either end of the full peptide. The functionalcomponent, and this linkage is made before, during, or after synthesisof the full peptide by coupling the appropriate molecules. For example,the inclusion of a cysteine or lysine residue in the full peptide allowsit to be attached to a functional component that contains anelectrophilic group such as a bromo or iodo group, or an ester oranhydride group, by means of the nucleophilic attack of the thiolsulphur atom of the cysteine residue or the amino nitrogen atom of thelysine residue on that electrophilic group of the functional component.Alternatively, a bifunctional cross-linking agent may be used to attachthe β-strand-forming section of peptide to the functional component; orthe full peptide may be synthesised using a specially preparedamino-acid derivative which already contains the functional component;or a standard coupling agent such as dicyclohexylcarbodiimide may beused to form an amide bond between a side-chain or terminal carboxyl oramino group of the peptide and an amino or carboxyl group of thefunctional component.

Uses of Peptides According to the Invention

The chemical compounds and compositions described herein can be used forany application which employs their ability to associate specificallywith target β-strands and thereby inhibit the association of otherβ-strands with those target β-strands. One application for thesecompounds is in their use to inhibit or reverse the aggregation ofproteins or peptides into insoluble β-fibres, or more specifically, toinhibit or reverse the association of β-strands into β-sheets, in vitroor in vivo. In vitro, for example, they can be used in combination withan additional agent such as urea, guanidinium chloride, or anotherdenaturant to assist in the refolding of denatured, misfolded, oraggregated proteins or peptides.

According to the present aspect of the invention, the denatured,misfolded, or aggregated protein or peptide is dialysed from a solutioncontaining the peptide according to the invention plus the additionalagent, for example, or by protein-renaturation chromatography through asolid matrix, resin, or support to which the peptide is covalentlyattached, in the presence of the additional agent.

The peptides are also useful in vivo or in vitro for the diagnosis,study, or treatment of diseases caused by the aggregation of proteins orpeptides into insoluble β-fibres, such as those listed in theintroduction. For such applications, the compounds are designed so thatthey can penetrate cell membranes and the blood-brain barrier, and sothat they are resistant to enzyme-catalysed proteolysis; a traceablegroup may also be incorporated into the invented compound as describedso that it may be used as a probe for the diagnosis of these diseases.

The peptides of the invention could also be used either in vitro or invivo to inhibit the oligomerisation or association of protein subunitswhere this occurs by the association of β-strands. Many enzymes andother proteins are active only as dimers or other oligomers which areformed from individual subunits by the association of β-strands, and theinvented compounds could be used to inhibit the activity of theseproteins by binding to these β-strands and thereby hindering theirassociation to form the complete protein complex. For example, thecatalytic activity of the HIV protease depends on its dimerisation,which involves the association of β-strands formed by its N- andC-terminal sections of peptide. Short peptides homologous to thesesections of peptide have been successfully used to inhibit thedimerisation and thereby the catalytic activity of this enzyme (Babe etal., 1992; Franciskovich et al., 1993; Schramm et al., 1993; Schramm etal., 1996; Schramm et al., 1992; Zutshi et al., 1997). These peptidesare, however, not very soluble in aqueous solutions and are susceptibleto degradation by proteolytic enzymes because they consist solely ofNα-unsubstituted α-L-amino-acid residues, therefore they are notsuitable for use as therapeutic agents. The compounds described hereinare more soluble in aqueous solutions and are resistant to degradationby proteolytic enzymes, so they are more suitable for use as therapeuticagents. For a review on the use of ‘interface’ peptides to inhibit theoligomerisation or association of protein subunits into activecomplexes, see reference (Zutshi et al., 1998).

Thus the ability of the peptides of the invention to inhibit theassociation of β-strands may be used for any application both in vitroand in vivo. In addition, the ability of these compounds to simplyassociate specifically with target β-strands may also be used as suchfor any in vitro or in vivo application. For example, the compoundscould be used as a traceable probe, especially as a histochemical stainor indicator, to indicate the presence or location of β-strands,β-sheets, or β-fibres in vitro or in vivo. In such applications, thecompound contains or is attached to an atom or group that contains aradioactive or magnetically active nucleus, or a fluorescent, coloured,or other spectroscopically detectable group such as a group whichcontains an unpaired electron and thereby acts as a spin label.Specifically, such a compound may be used as a histochemical stain orindicator to monitor the production of insoluble β-fibres in patients ofAlzheimer's Disease and other neurodegenerative diseases caused by theaggregation of proteins or peptides into insoluble β-fibres in thebrain.

The peptides according to the invention may be attached to a solidmatrix, resin, or support and used as such for protein-renaturationchromatography as described above; they could also be used in this formfor affinity chromatography wherein the β-strand-forming section ofpeptide acts as a bait to capture the proteins or peptides which formthe target β-strand. For example, a β-strand-forming section of peptidedesigned to form a β-strand and associate specifically as such with atarget β-strand formed by a particular protein of biochemical interestcould be attached to a solid matrix, resin, or support to enablepurification of that particular protein by affinity chromatography: theprotein which contains the target β-strand will bind to the β-strandformed by the β-strand-forming section of peptide attached to the solidsupport, and may thereby be separated from other proteins which will notbe recognised by the β-strand-forming section of peptide; the purifiedprotein may then be liberated from the support by adding a free form ofthe β-strand-forming section of peptide, or some other agent whichdisrupts the interaction between the two β-strands, such as urea or someother denaturant.

Finally, the compounds described herein may be included in acombinatorial library of such compounds to screen for one particularcompound which is to be used for any of the above applications. Thiscombinatorial library could be prepared by any suitable standard methodof preparing synthetic peptide libraries (Lebl and Krchnak, 1997),wherein Nα-substituted α-D-amino acid residues are included in thepeptides at appropriate positions according to the present invention.The resulting library is then screened for peptides which bind to atarget β-strand sufficiently tightly, or which sufficiently inhibit theactivity of an oligomeric protein by blocking its oligomerisation, orwhich rescue cells that would otherwise be killed by the aggregation ofproteins or peptides into insoluble β-fibres. The selected compounds maybe used directly for any of the above applications, or used to designcombinatorial libraries of compounds which are even more active, orwhich are more suitable for use as therapeutic agents.

For use as therapeutic agents, the peptides according to the inventionmay be formulated according to established practices. The peptideaccording to the invention may be administered in a convenient mannersuch as by the oral, intravenous (where water soluble), intramuscular,subcutaneous, intranasal, intradermal or suppository routes orimplanting (e.g. using slow release molecules). Depending on the routeof administration, the peptide may be required to be coated in amaterial to protect it from the action of enzymes, acids and othernatural conditions which may inactivate it.

In order to administer the peptide by other than parenteraladministration, it may be coated by, or administered with, a material toprevent its inactivation. For example, the peptide may be administeredin an adjuvant, co-administered with enzyme inhibitors or in liposomes.Adjuvant is used in its broadest sense and includes any immunestimulating compound such as interferon. Adjuvants contemplated hereininclude resorcinols, non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecyl polyethylene ether. Enzyme inhibitors includethose of pancreatic trypsin and other digestive proteases.

Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes.

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene gloycol, and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of superfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the sterilised active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze-drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

When the peptide is suitably protected as described above, it may beorally administered, for example, with an inert diluent or with anassimilable edible carrier, or it may be enclosed in hard or soft shellgelatin capsules, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. The amountof active compound in such therapeutically useful compositions is suchthat a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac, sugar or both. A syrup orelixir may contain the active compound, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavouring such ascherry or orange flavour. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such as active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredients are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The present invention provides the use of a peptide according to theinvention for the manufacture of a medicament for the treatment ofdisease associated with aberrant protein/polypeptide structure. Theaberrant nature of the protein/polypeptide may be due to misfolding orunfolding which in turn may be due to an anomalous e.g. mutatedamino-acid sequence. The protein/polypeptide may be destabilised ordeposited as plaques e.g. as in Alzheimer's disease. The disease mightbe caused by a prion. A polypeptide-based medicament of the inventionwould act to renature or resolubilise or inhibit the accumulation ofaberrant, defective or deposited proteins.

The invention is further described, for the purposes of illustrationonly, in the following examples.

EXAMPLE 1

Aggregation of the Alzheimer's Aβ peptide into amyloid fibres is causedby the intermolecular association of five-residue KLVFF (SEQ ID NO: 1)peptide segments comprising residues 16–20 of the Aβ peptide (Tjernberget al., 1997). A peptide, referred to below as Peptide X (SEQ ID NO: 2),was therefore constructed to associate tightly with the KLVFF motif (SEQID NO: 1), in order to inhibit aggregation of the Aβ peptide.

The sequence of side chains in Peptide X is LLLLRR (SEQ ID NO: 2), whichis highly homologous to the reverse sequence FFVLK (SEQ ID NO: 3),except that an additional residue having an arginine side chain has beenadded to the C-terminus.

Leucine side chains were selected to take the place of all fourhydrophobic side chains in the FFVLK sequence (SEQ ID NO: 3) becausethey are relatively flexible and can adapt their conformation to makestrong hydrophobic interactions with the neighbouring hydrophobic sidechains of an associated β-strand, while an arginine side chain waschosen to take the place of the lysine side chain in the FFVLK sequence(SEQ ID NO: 3) because it can form a stronger electrostatic interactionwith one of the two acidic side chains which follow theaggregation-causing KLVFF (SEQ ID NO: 1) segment of the target β-strand.

The additional residue having an arginine side chain at the C-terminusof Peptide X (SEQ ID NO: 2) may form another strong electrostaticinteraction with the second of these two acidic side chains, and shouldfurther assist Peptide X (SEQ ID NO: 2) to penetrate cell membranes andthe blood-brain barrier.

Finally, the N-terminal amino group of Peptide X (SEQ ID NO: 2) wasacetylated to maximise its association with the aggregation-causingKLVFF segment (SEQ ID NO: 1) of the target β-strand, and its otherwisenegatively charged C-terminal carboxyl group was amidated to furtherimprove the ability of Peptide X (SEQ ID NO: 2) to penetrate cellmembranes and the blood-brain barrier. In this way, Peptide X (SEQ IDNO: 2) has been designed to associate specifically as a β-strand withthe aggregation-causing KLVFF (SEQ ID NO: 1) segment of the targetβ-strand formed by the Alzheimer's Aβ peptide to form an parallelβ-sheet complex, thereby sterically hindering the aggregation of the Aβpeptide into insoluble β-fibres.

Peptide X (SEQ ID NO: 2) is a substituted peptide, in accordance withthe present invention. The sequence, including substituents, isNα-acetyl-(D-leucine)-(Nα-methyl-D-leucine)-(D-leucine)-(Nα-methyl-D-leucine)-(D-arginine)-(D-arginine)-NH₂,or all-D-[Ac-Leu-meLeu-Leu-meLeu-Arg-Arg-NH₂.

Peptide X (SEQ ID NO: 2) was synthesised by 9-fluorenylmethoxycarbonyl-(Fmoc-) based solid-phase peptide synthesis (Fields and Noble, 1990)using the coupling agent 1-hydroxy-7-azabenzotriazole (HOAt), which isable to couple sterically hindered amino-acid residues (Angell et al.,1994; Carpino et al., 1994).

Peptide X (SEQ ID NO: 2) was found to be completely soluble in aqueoussolutions over a wide range of pH values, even at a concentration of 10mM (about 10 mg/ml); yet, except for the two positively charged arginineside chains, it is extremely hydrophobic and is therefore able topenetrate cell membranes and the blood-brain barrier, especially as itis only six amino-acid residues in length. The two positively chargedarginine side chains assist the peptide to penetrate cell membranes andthe blood-brain barrier by making electrostatic interactions with thenegatively charged phosphate head groups of their constituentphospholipid molecules, resulting in the formation of inverted micelleswhich carry the peptide molecules across these membranes.

The capacity of Peptide X (SEQ ID NO: 2) to inhibit the aggregation of asynthetic peptide fragment corresponding to residues 11 to 25 of theAlzheimer's Aβ peptide into amyloid fibrils was determinedquantitatively using a standard assay based on the amyloid-dependentfluorescence of thioflavin T at 482 nm (Levine, 1993).

Peptide X (SEQ ID NO: 2) was dissolved in water to a concentration of 10mM (about 10 mg/ml). Alzheimer's Aβ peptide fragment, at a concentrationof 50/M (about 0.1 mg/ml) in 50 mM sodium acetate buffer (pH 5.0), wasincubated at 25° C. in the absence or presence of Peptide X (SEQ ID NO:2) at concentrations ranging from 100 μM to 1 mM; the aggregation of theAβ peptide fragment into insoluble β-fibres in the solutions wasdetermined quantitatively after 20 minutes by measuring the fluorescenceof 1%M added thioflavin T at 482 nm using an excitation wavelength of440 nm. 5 ml aliquots of these solutions were then analysed by electronmicroscopy to confirm that Peptide X (SEQ ID NO: 2) had inhibited and/orreversed the aggregation of the Alzheimer's Aβ peptide fragment intoinsoluble β-fibres.

According to this assay, the aggregation of the Aβ peptide fragment intoamyloid fibrils was inhibited by more than 60% in the presence of 200 μMPeptide X (SEQ ID NO: 2) (see FIG. 5). Similar results were obtainedwhen Peptide X (SEQ ID NO: 2) was added to the Aβ peptide fragment afterincubation, showing that Peptide X (SEQ ID NO: 2) is able todisaggregate preformed amyloid fibrils. Analysis of the Aβ peptidefragment incubated with and without 500 mM Peptide X (SEQ ID NO: 2) byelectron microscopy confirmed that Peptide X (SEQ ID NO: 2) had almostcompletely inhibited aggregation of the Aβ peptide fragment into amyloidfibrils (see FIGS. 6 and 7).

FIGS. 3 and 4 show how Peptide X (SEQ ID NO: 2) forms a β-strand (X) andassociates as such with one edge of a target β-strand (Y) formed by asegment of the Aβ peptide or some other peptide-based molecule in eitherorientation to form a parallel (FIG. 3) or antiparallel (FIG. 4)two-stranded β-sheet complex, thereby sterically hindering theassociation of other β-strands with that edge of the target β-strand.

The entire length of Peptide X (SEQ ID NO: 2) is able to form a β-strandbecause it consists solely of α-D-amino acid residues which stericallypermit it to do so: they are all able to adopt the respective phi andpsi angles required to form a β-strand. Furthermore, the stericconstraints imposed by the Nα-methyl groups of the twoNα-methyl-α-D-amino acid residues (residues 2 and 4) serve to encouragePeptide X (SEQ ID NO: 2) to form a β-strand. When Peptide X (SEQ ID NO:2) does form a β-strand, these two Nα-methyl groups lie along the sameedge of the β-strand, as shown in either FIG. 3 or FIG. 4, because theyare an even numbers of residues (in this case two residues) apart fromeach other and the repeating unit of a β-strand is two residues. Thisedge of the β-strand formed by Peptide X (SEQ ID NO: 2) is stericallyhindered by these two Nα-methyl groups from associating with anotherβ-strand. The other edge of the β-strand formed by Peptide X (SEQ ID NO:2), however, remains free to do so, and can associate in either theparallel or antiparallel orientation with a free edge of a targetβ-strand formed by a segment of the Aβ peptide or some other protein orpeptide molecule to form a parallel (FIG. 3) or antiparallel (FIG. 4)two-stranded β-sheet complex, thereby sterically hindering theassociation of other β-strands with that edge of the target β-strand,and thus preventing the formation of extended β-sheets and thedeposition of insoluble pathogenic β-fibres. This association of theβ-strand formed by Peptide X (SEQ ID NO: 2) with the target β-strand ismade by hydrogen bonds between their backbone peptide groups andadditional non-covalent interactions between their side chains.

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1. A peptide comprising (a) said peptide comprises a β-strand-formingsection, said section consisting of four to sixteen consecutiveα-D-amino residues and encompassing at least 50% of the length of saidpeptide; (b) each of the consecutive α-D-amino acid residues in saidβ-strand-forming section has a side chain; (c) said β-strand-formingsection forms a β-strand having a peptide backbone which takes on theform of an extended ribbon having two edges, a first edge whichassociates with a target β-strand formed by a separatepeptide-containing molecule and a second edge, such that the NH and COcomponents of successive α-D-amino acid residues lie along the firstedge and the second edge of the ribbon, the first edge and second edgecorresponding to two opposite edges of the plane of the ribbon, and theside chains of the consecutive α-D-amino acid residues beingalternatively above or below the plane of the ribbon; (d) at least oneof the Nα-atoms within the peptide backbone of the β-strand is aNα-substituted with an Nα-substituent, such that one or moreNα-substituent lie along only the second edge and sterically hinders theassociation of the second edge with another β-strand; and (e) the firstedge remains free of Nα-substituents, and is not prevented fromassociating with the target β-strand formed by the separatepeptide-containing molecule.
 2. The peptide according to claim 1,wherein, when there are two or more successive Nα-substituted α-D-aminoacid residues, no two successive Nα-substituted α-D-amino acid residuesin the β-strand-forming section are separated by more than 3 consecutiveNα-unsubstituted α-D-amino acid residues.
 3. The peptide according toclaim 1 wherein, when there are two or more successive Nα-substitutedα-D-amino acid residues, the successive Nα-substituted α-D-amino acidresidues in the β-strand-forming section are separated from each otherby single Nα-unsubstituted α-D-amino acid residues, such that theβ-strand-forming section comprises an alternating sequence ofNα-substituted and Nα-unsubstituted α-D-amino acid residues.
 4. Thepeptide according to claim 1 wherein the Nα-substituent of eachNα-substituted α-D-amino acid residue in the β-strand-forming sectionsterically allows or promotes the β-strand-forming section to form aβ-strand, and sterically hinders the association of said second edge ofthat β-strand with any other β-strand.
 5. The peptide according to claim4, wherein the Nα-substituent of each Nα-substituted α-D-amino acidresidue in the β-strand-forming section is selected from the groupconsisting of: a fluorine atom or an OH group; a group that is connectedto the Nα atom by an oxygen atom within it; a group that is connected tothe Nα atom by a CH₂ subgroup within it; a methyl or ethyl group, orsome other alkyl or aliphatic group; a substituted or unsubstitutedbenzyl group, or some other arylmethyl group; an acetylated or acylated2-hydroxy-4-methoxybenzyl (AcHmb) group; and an acylated or unacylated2-hydroxybenzyl (AcHb/Hb) group.
 6. The peptide according to claim 1,wherein the side chain of each α-D-amino acid residue in theβ-strand-forming section allows or promotes the α-strand forming sectionto form a β-strand.
 7. The peptide according to claim 6, wherein theside chain of one or more α-D-amino acid residues in theβ-strand-forming section is that of an amino acid residue having aβ-sheet propensity of greater than 1.00.
 8. The peptide according toclaim 6, wherein the side chain of any one or more α-D-amino acidresidues in the β-strand-forming section is selected from the groupconsisting of: an atom or group that allows or promotes theβ-strand-forming section to associate as a β-strand with the targetβ-strand and thereby form a stable β-sheet complex; and an atom or groupthat forms a hydrophobic or electrostatic interaction, hydrogen bond, orother favourable non-covalent interaction with the neighbouring sidechain of the target β-strand in a β-sheet complex comprising the targetβ-strand and the β-strand-forming section.
 9. The peptide according toclaim 6, wherein the side chain of any one or more α-D-amino acidresidues in the β-strand-forming section is selected from the groupconsisting of: a hydrophobic group, or a group that has a considerablehydrophobic portion; a branched or unbranched alkyl or aliphatic group;a group that is branched at its connecting β-carbon atom; an aromaticgroup; an acidic or basic group; and an amide- or hydroxyl-containinggroup.
 10. The peptide according to claim 1, wherein the side chain ofone or more α-D-amino acid residues in the β-strand-forming sectionhinders stacking of β-sheets.
 11. The peptide according to claim 10,wherein the side chain of one or more α-D-amino acid residues in theβ-strand-forming section extends beyond the neighbouring side chains inthe β-strand.
 12. The peptide according to claim 1, wherein the sidechain of one or more α-D-amino acid residues in the p-strand-formingsection contains a detectable group which allows the peptide to betraced or detected.
 13. The peptide according to claim 12, wherein theside chain of one or more α-D-amino acid residues in theβ-strand-forming section is selected from the group consisting of: anatom or group that contains a radioactive or magnetically activenucleus; that of phenylalanine or tyrosine with one or more radioactiveor magnetically active iodine or other halogen atoms substituted ontothe aromatic ring; a fluorescent, coloured, or other spectroscopicallydetectable group; a group which contains an unpaired electron andthereby acts as a spin label; a group which contains the2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL) group; and a group whichcontains the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) group.
 14. Thepeptide according to claim 1, wherein the side chain of one or moreα-D-amino acid residues in the β-strand-forming section is selected fromthe group consisting of the side chain of: any naturally occurringα-L-amino acid or synthetic derivative thereof; alanine; serine;cysteine; threonine; valine; leucine; isoleucine; methionine;phenylalanine; tyrosine; tryptophan; glutamine; asparagine; glutamate;aspartate; histidine; lysine; arginine; and tert-leucine orβ-hydroxyvaline.
 15. The peptide according to claim 1 wherein the targetp-strand is formed by the Alzheimer's Aβ peptide, and theβ-strand-forming section binds specifically as a β-strand to part or allof the KLVFFAE sequence (SEQ ID NO: 51 within the target α-strand in theparallel orientation, thereby forming a parallel β-sheet complex whereinconsecutive residues of the β-strand-forming section lie directlyopposite consecutive residues of SEQ ID NO:5 in the same order.
 16. Thepeptide according to claim 1 wherein the target β-strand is formed bythe Alzheimer's Aβ peptide, and the β-strand-forming section bindsspecifically as a β-strand to part or all of the KLVFFAE sequence (SEQID NO: 5) within the target β-strand in the antiparallel orientation,thereby forming an antiparallel β-sheet complex wherein consecutiveresidues of the β-strand-forming section lie directly oppositeconsecutive residues of SEQ ID NO:5 in reverse order.
 17. The peptideaccording to claim 1 wherein the β-strand-forming section is precededby, or followed by, or otherwise attached to a distinctmembrane-penetrating section of the peptide which enables theβ-strand-forming section to cross cell membranes, the blood-brainbarrier or any other biological barrier.
 18. The peptide according toclaim 17 wherein the side chain of each residue in themembrane-penetrating section is selected from the group consisting of: abasic or hydrophobic group; and a side chain of alanine, valine,leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan,proline, histidine, lysine, or arginine.
 19. The peptide according toclaim 1 wherein the β-strand-forming section has a free or acylated Nterminus and a free, or amidated, or esterified C terminus, or formspart of a larger peptide which has a free or acylated N terminus and afree, amidated, or esterified C terminus.
 20. The peptide according toclaim 1 wherein the β-strand-forming section is attached to anotherfunctional component.
 21. The peptide according to claim 20, wherein thefunctional component is selected from the group consisting of: acomponent which strengthens the binding of the β-strand-forming sectionto the target β-strand; a component which enhances specificity ofassociation of the β-strand-forming section with the target β-strand; acomponent which enables the β-strand-forming section to cross cellmembranes, the blood-brain barrier and other biological barrier; acomponent which causes the peptide to target specific organs, cells, ormolecules; a component which allows the peptide to be traced ordetected; an atom or group that contains a radioactive or magneticallyactive nucleus; a fluorescent, coloured, or other spectroscopicallydetectable group; a group which contains an unpaired electron andthereby acts as a spin label; a group which contains the 2, 2, 5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL) group or the 2, 2, 6,6-tetramethyl-1-piperidinyloxy (TEMPO) group; a solid matrix, resin, orsupport; an enzyme, hormone, antibody, transcription factor, or otherprotein molecule; a group that binds specifically to a particularprotein; and a cytotoxic molecule.
 22. The peptide according to claim20, wherein attachment of the β-strand-forming section to the functionalcomponent is by means of: an amide or ester linkage formed with theC-terminus of the β-strand-forming section; or an amide linkage formedwith the N-terminus of the β-strand-forming section; or an amide linkageformed with a carboxyl, or amino group of a side chain within theβ-strand-forming section; or an ester linkage formed with a carboxyl orhydroxyl group of a side chain within the β-strand-forming section; or adisulphide bridge formed with a thiol group of a side chain within theβ-strand-forming section.
 23. The peptide according to claim 1 whereinthe β-strand-forming section comprises between 5 and 10 amino acidresidues and/or includes side chains of amino acid residues of theβ-strand-forming section that are homologous to or identical to theamino-acid sequence FFVLK of SEQ ID NO:
 3. 24. The peptide according toclaim 1 wherein the β-strand-forming section associates with a targetβ-strand comprising the amino-acid sequence KLVFF of SEQ ID NO:
 1. 25.The peptide according to claim 1 comprising one or more components whichmimic the structure and action of said β-strand-forming section, whereinthe components which mimic the structure and action of theβ-strand-forming section are formed by replacing one or more of thebackbone peptide groups or side-chain groups of amino acid residues ofthe β-strand-forming section by another chemical group of similarstereochemistry and ability to form favourable non-covalent interactionswith the target β-strand.
 26. The peptide according to claim 25 wherein:(a) one or more of the Nα-unsubstituted backbone peptide groups (CONH)of the β-strand-forming section is/are each replaced by any of thefollowing groups: CSNH (thioamide); COO (ester); CSO or COS (thioester);CSS (dithioester); COCH₂ (ketone); CSCH₂ (thioketone); SO₂NH(sulphonamide); SOCH₂ (sulphoxide); SO₂CH₂ (sulphone); SO₂O(sulphonate); and/or (b) one or more Nα-substituted backbone peptidegroups (CON(R)) of the β-strand-forming section is/are replaced by oneof the following N- or C-substituted groups: CSN(R) (thioamide); COCH(R)(ketone); CSCH(R) (thioketone); SO₂N(R) (sulphonamide); SOCH(R)(sulphoxide); SO₂C(R) (sulphone), wherein R is equivalent to theoriginal Nα-substituent; and/or (c) one or more of the side chains ofthe β-strand-forming section is/are each replaced by another grouphaving stereochemistry or arrangement of polar and non-polar atoms,similar to that of the replaced side chains, maintaining thoseparticular features which are essential for association with the targetβ-strand.
 27. The peptide ording to claim 1, wherein any two successiveNα-substituted α-D-amino acid residues are separated by an odd number ofconsecutive Nα-ubstituted α-D-amino acid residues.
 28. A pharmaceuticalcomposition comprising the peptide according to claim 1.